Cells were lysed in RIPA buffer. Total protein concentration was determined by DC™ Protein Assay kit (Bio-Rad). A total of 30 µg of protein was separated using 4-15% gradient SDS-PAGE (4-15% Ready Gel® Tris-HCL Gel; Bio-Rad) and transferred onto a PVDF membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was probed with primary antibodies that specifically recognized HA (mouse anti-HA.11 clone 16B12 monoclonal 1:1,000; Covance) and α-tubulin (mouse anti-α-tubulin clone DM1A monoclonal; Sigma). The membrane was then incubated with secondary antibody (HRP-conjugated goat anti-mouse 1:10,000; Abcam) after thorough washing and visualized with a Western Lighting™ Ultra Chemiluminescence Substrate kit (PerkinElmer) using the ChemiDoc™ XRS+ system (Bio-Rad). The quantification of MagA-HA was performed using ImageLab™ software (Bio-Rad) and was normalized to α-tubulin.
Hrp conjugated goat anti mouse
Manufactured by Abcam
Sourced in United States
HRP-conjugated goat anti-mouse is a secondary antibody that is conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to mouse primary antibodies in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Abcam (2 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Ready gel tris hcl gel, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Western lighting ultra chemiluminescence substrate kit, by PerkinElmer (1 mentions)
Mouse anti ha 11 clone 16b12 monoclonal, by Fortrea (1 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protease inhibitor, by Cell Signaling Technology (1 mentions)
E cadherin, by Abcam (1 mentions)
N cadherin, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Cleaved caspase 3, by R&D Systems (1 mentions)
Rabbit anti rat igg, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Hrp conjugated goat anti rabbit antibody, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
9 protocols using hrp conjugated goat anti mouse
1
Quantitative Western Blot Analysis of MagA-HA
2
Western Blot Analysis of Protein Markers
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Cells were lysed in lysis buffer containing protease inhibitors (Cell Signaling, USA). BCA protein assay reagent (Thermo Fisher Scientific, USA) was used to determine the concentration of the isolated proteins. 25 μg of the protein was fractioned by SDS-PAGE and afterwards transferred to a PVDF membrane (EMD Millipore, USA). The membrane was then incubated with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 1 h. The membrane was washed once with TBST, followed by incubation with primary antibodies against MDR1 (Novus Biologicals, USA) (1:1000), α-enolase (Boster, China) (1:1000), XIAP (Abcam, USA) (1 μg/mL), cleaved caspase-3 (R&D systems, USA) (0,5 μg/mL), E-Cadherin (1:500), N-Cadherin (1:1000) (Abcam, USA), and β-actin (1:10,000) (Sigma Aldrich, USA). The membrane was then washed three times for 5 mins with TBST on a shaker and incubated with HRP-conjugated goat anti-mouse (1:10,000) or goat anti-rabbit (1:10,000) IgG secondary antibodies (Abcam, USA) and visualized via Immobilon Western Chemiluminescent HRP substrate (EMD Millipore, USA). Densitometry of blots was analyzed using Image J.
3
Quantifying Antigen-Specific Antibody Titers by ELISA
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Antibody titers were determined by ELISA as described previously (17 (link), 42 (link)). Briefly, each well of a 96-well plate was coated with 100 ng of F1mutV or PA diluted in coating buffer (0.05 M sodium carbonate-sodium bicarbonate [pH 9.6]) overnight at 4°C. The plates were then blocked with 3% bovine serum albumin (BSA) in PBS (pH 7.4) for 1 h at 37°C. After 1 h of incubation at 37°C with serially diluted serum samples, plates were washed with PBS-T (PBS with 0.1% Tween 20 [pH 7.4]). For total IgG, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (KPL, Gaithersburg, MD), rabbit anti-rat IgG (Invitrogen), or goat anti-rabbit IgG (KPL, Gaithersburg, MD) was used as the secondary antibody. For mouse or rat IgG subtypes, HRP-conjugated goat anti-mouse or HRP-conjugated mouse anti-rat IgG1 or IgG2a secondary antibodies (Abcam, Cambridge, MA) were used. Samples were initially diluted 1:200; serial fivefold dilutions were performed as necessary to ensure that values reached the endpoint. For rabbit anti-PA IgG titers, plates were coated with PA, and affinity-purified rabbit anti-PA polyclonal antibody was used to generate a standard curve, from which the sample anti-PA IgG concentrations (in nanograms per milliliter) were determined. 3,3′,5,5′-Tetramethylbenzidine (TMB) microwell peroxidase substrate kit (KPL, Gaithersburg, MD) was used for staining.
4
Western Blot Analysis of FN1 and SRSF3
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Cells were harvested and lysed on ice with RIPA buffer (Beyotime, China). Next, the lysate was incubated on ice for 15 min and then centrifuged for 15 min (13,000 rpm, 4 °C). The supernatant was collected, and the protein concentration was determined by using the BCA Protein Assay Kit (Beyotime, China). Protein samples (20 μg) were loaded into each lane of the SDS-PAGE lane. Following electrophoresis, proteins were transferred to PVDF membranes blocked with 3% BSA. Protein transferred membranes were incubated with primary antibodies overnight at 4 °C, followed by 1 h of incubation with secondary antibodies at room temperature. Western blots were visualized on a BioSpectrum 515 (Wolf Laboratories Limited, UK) by using the Affinity ECL kit (Affinity, china). The antibodies used in the western blot were: FN1 (1:1000 dilution, Abcam, USA), SRSF3 (1:300 dilution, Santa Cruz Biotechnology, USA), GAPDH (1:5000 dilution, Abcam, USA), HRP-conjugated goat anti-mouse (1:5000 dilution. Beyotime, China), and HRP-conjugated goat anti-rabbit antibody (1:5000 dilution, Beyotime, China).
5
Protein Analysis of Tick Tissues
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Tick guts and salivary glands were dissected in cold PBS and lysed with RIPA buffer supplemented with 1 mM PMSF. The supernatant was collected after centrifugation at room temperature (25°C), at 14,000 × g for 10 min. The supernatants obtained were used as the samples for Western blotting. Samples were electrophoresed for 1 h 30 min at 100 V constant current. The SDS-PAGE gel was transferred to a PVDF membrane for 1 h at 100 V. The membrane was blocked with 10% skimmed milk in TBST for 5 h at room temperature (25°C), followed by three washes with TBST. It was then incubated overnight with purified anti-GST-Bscp31 antibody (prepared in the laboratory) diluted to 1:500 in TBST. Following this, the membrane was washed three times with TBST and incubated in secondary antibodies (HRP-conjugated goat anti-mouse; Abcam; 1:2000 dilution) for 2 h, in the dark at room temperature (25°C). Protein bands in the membrane were visualized after staining with enhanced chemiluminescence solution.
6
Protein Extraction and Quantification
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Cells were lysed with RIPA buffer supplemented with protease inhibitor (Roche, Basel, Switzerland). Both nuclear and cytoplasmic proteins were extracted and separated using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Protein concentrations were determined by bicinchoninic acid assay (BCA) assay (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts (20 µg) of protein were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 3% BSA-TBST for 1 hour, the membrane was incubated with primary antibody at 4°C overnight. The following primary antibodies were used: rabbit anti-β-catenin (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-WISP1 (1:1000, Abcam, Cambridge, UK), mouse anti-H2A (1:1000, Cell Signaling Technology), and mouse anti-GAPDH (1:5000, Abcam). The following secondary antibodies were used (1:10,000): horseradish peroxidase (HRP)-conjugated goat‐anti-mouse (Abcam) and HRP‐goat‐anti-rabbit (Abcam). Proteins were detected using an Odyssey infrared scanner (Li-Cor Biosciences, Lincoln, NE, USA). The band intensities were estimated using Photoshop Software.
7
Western Blot Analysis of RUNX1, p38, and VEGF
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For Western blot analysis, HRMECs and tissue samples were lysed in RIPA buffer containing protease inhibitor cocktail (Generay, Shanghai, China). The proteins were transferred to PVDF membranes and probed with primary antibodies, including anti-RUNX1 (#ab35962; dilution, 1:1000), anti-p38 (#ab170099; dilution, 1:1000), anti-VEGF (#ab222510; dilution, 1:1000) and anti-GAPDH (#ab181602; dilution, 1:1000) or anti-H3 (#ab1791; dilution, 1:1000); all primary antibodies were purchased from Abcam (Cambridge, MA, U.S.A.). The second antibody HRP-conjugated goat anti-mouse (#ab19195; dilution, 1:10,000) and goat anti-rabbit IgG (#ab6721; dilution, 1:10,000) were used to detect the expression of T-RUNX1, T-p38, T-VEGF and T-GAPDH, respectively; all secondary antibodies were purchase from Abcam (Cambridge, MA, U.S.A.). The blots were detected using Bio-Imaging System and Quality One 1-D analysis software (Bio-Rad, Richmond, CA, U.S.A.).
8
Western Blot Analysis of Protein Biomarkers in Parkinson's Disease
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The protein was extracted from the SNpc tissue by RIPA lysis buffer (cat. no. R0278; Sigma-Aldrich). After centrifugation at 12,000 rpm for 20 min at 4°C, total proteins were harvested. The bicinchoninic acid assay (BCA; cat. no. 23225; Thermo Fisher Scientific) was used to determine the concentration of total proteins. The total protein (30 ug) was separated with 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (PVDF; Millipore). After blocking with 5% BSA for 2 h, the membrane was incubated with primary antibodies (TH, 1 : 1000, cat. no. ab152, Millipore; GPX4, 1 : 1000, cat. no. ab125066, Abcam; FTH1, 1 : 1000, cat. no. ab183781, Abcam; GAPDH, 1 : 5000, cat. no. ab8245, Abcam) at 4°C overnight. The membrane was then incubated with HRP-labelled secondary antibodies (horseradish peroxidase- (HRP-) conjugated goat anti-rabbit, 1 : 5000, cat. no ab6721, Abcam; HRP-conjugated goat anti-mouse, 1 : 5000, cat. no. ab6789, Abcam) for 1 hour at room temperature. The ECL substrate (Pierce, Thermo Fisher Scientific, Inc. USA) was used to visualize the bands. The data were analyzed by image j.
9
Immunoprecipitation and Western Blot Analysis
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Immunoprecipitates were resuspended in reducing buffer and heated to 100°C for 6 min. Then, they were loaded and resolved on an 8% SDS-PAGE, and transferred to a PVDF membrane. The blots were probed with mAb against SLex [clone KM93, Calbiochem; diluted 1/67 in TBST buffer (Tris-HCl 10 mM pH 7.5, NaCl 100 mM, 0.1% Tween 20) containing 0.5% BSA)]; or with fluorescein conjugated SNA lectin [Vector Laboratories; diluted 1/1000 in lectin buffer (150 mM NaCl, 0.1 M Tris-HCl pH 7.5, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2)] for 2 h at room temperature. After three washes with TBST, membranes were incubated with secondary antibody HRP-conjugated goat anti-mouse (Abcam, UK; diluted 1/40000 in TBST-0.5% BSA); or sheep anti-fluorescein (Roche Diagnostics, diluted 1/2500 in TBST-1% BSA), respectively, for 1 h at room temperature. Immunoreactive bands were visualized using Immobilon Western Chemiluminescent HRP Substrate kit (EMD Millipore Corporation; MA, USA). Equal amounts of loaded α2 integrin were corroborated by stripping the membranes and blotting with HRP-conjugated streptavidin (GE Healthcare; diluted 1/100000 in TBST-1% BSA). Two independent experiments were performed.
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